Dihydrofolate reductase is to be purified from a number of sources: isoenzymes from S. faecium, isoenzymes from rat liver, beef liver and Neisseria gonorrhoeae. The enzyme will be purified in each case by a combination of classical methods and affinity chromatography. The structure of the reductase from the various sources will be examined by a number of methods including sequence determination, and X-ray diffraction studies where suitable crystals can be obtained. Chemical modification studies will be performed to obtain evidence of residues at the catalytic site and at binding sites for substrates and inhibitors. The bacterial strains will be grown under conditions in which amino acids specifically labeled with 13C are incorporated into the reductase. 13C-NMR will then be used to investigate the state of mobility of the residues, to monitor conformational states of the enzyme in the presence and absence of ligands and to obtain evidence of direct interactions of substrates and inhibitors with residues. The results of this work will be used to describe the stereochemical fit of inhibitors to reductase from various sources. This information will be used for the prediction of more selective inhibitors.